Microorganism culture device and microorganism culture medium comprising the same

ABSTRACT

A microorganism culture device comprising a porous matrix layer having a basis weight of 40 to 100 g/m 2  and an air permeability of 7 to 24 cm/sec, and at least one layer of a water-soluble polymer layer laminated with the matrix layer.

This application is a divisional of U.S. patent application Ser. No.10/938,296, filed 10 Sep. 2004 (now U.S. Pat. No. 7,312,072), which is aContinuation of U.S. patent application Ser. No. 10/168,250, filed 17Jun. 2002 (now abandoned), which is a National Stage of InternationalPatent Application No. PCT/JP00/08923, filed 15 Dec. 2000, and whichapplication(s) are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a microorganism culture device forculturing and detecting microorganisms in foods and in environmentalmonitoring, and to a microorganism culture medium comprising the same.Further, the invention relates to a sheet-form microorganism culturedevice and a sheet-form microorganism culture medium comprising thesame.

BACKGROUND ART

Microorganism culture tests, which have been performed in order to, forexample, determine a total aerobic count in food test, haveconventionally comprised the following steps: dissolving agar powder forpreparing a culture medium; sterilizing the medium; storing the mediumat approximately 45° C. to avoid solidification; injecting a certainvolume of the medium into a sterilized petri dish containing a certainvolume of a sample solution (for example, a suspension of a food);solidifying the agar after mixing; culturing at constant temperature;and counting the number of colonies of microorganisms. This conventionalmethod of microorganism culturing involves not only preparing a culturemedium in advance and sterilizing the medium, but also storing themedium at a sufficiently high temperature to avoid solidification,thereby requiring much time and labor.

Demand exists for omitting these time-consuming preparation steps inorder to conduct simple and rapid microorganism culture test. Inaddition, glass and plastic petri dishes used for the tests involveproblems. In food tests, which require numerous tests of microorganismculturing, cleaning and sterilizing glass petri dishes for reuse arevery time consuming. Disposable sterilized plastic petri dishes haverecently come into wide used, raising another problem of handling of alarge volume of plastic wastes. For the determination of total aerobiccounts in an environmental microorganism test, a conventional methodincludes the steps of wiping a certain area of a test subject with apiece of gauze or swab; rinsing the gauze or swab in sterilized water orsaline to thereby obtain a suspension of the microorganisms adsorbed onthe gauze or swab; applying the suspension on a prepared agar medium ormixing and diluting the suspension with an agar medium as describedabove; culturing the microorganisms; and counting microorganismcolonies. This method is also very time- and labor-intensive.

In order to solve those problems, simplified culture media which do notrequire preparation processes have been studied, manufactured, andcommercially distributed. These simplified culture media may beclassified into 4 types: a press type, a filter type, a film type, and atest paper type.

Japanese Patent Application Laid-Open No. 4-117299 discloses an testmethod using a press-type medium, which comprises the steps ofdispensing an agar medium into a plastic container having a mound-likeshape; and bringing the medium into direct contact with a test subject.The method is simple and useful for testing microorganism contaminationin an environment; however, it cannot provide quantitative results,because the medium has a small surface area, and difficult to employedto test subjects having a curved or uneven surface, thereby making themethod inapplicable to conventional food tests. Like the conventionalmethod, the method also involves a problem of generating a lot ofplastic waste after culturing, because plastic containers are used.

ANSI/ASTM F 488-79 describes a test method using a filter-type medium,which is typically a water-absorbent substance containing nutrients andcovered with a membrane filter, the method comprising immersing theculture medium in a sample solution for impregnation; catchingmicroorganisms on the filter surface; and culturing the microorganisms.The filter-type medium is suitable for liquid samples, but encountersdifficulty in test of solid-containing samples.

In a known test method using a test-paper-type medium, a filter paper isimpregnated with nutrients for promoting growth of microorganisms, and asample solution is added to or absorbed by the filter paper, which isthen stored in a plastic bag for culturing. However, the paper mediumcannot provide quantitative results, because cultured colonies arelikely to disperse and microorganisms grown inside the paper aredifficult to observe.

Japanese Patent Publication No. 2-49705 and Application Laid-Open No.3-015379 disclose test methods employing film-type media. These methodsuse a laminate of two films sandwiching a nutrient and a gelling agent,which is reconstituted to a gel when cold water is added thereto.Subsequently, microorganisms are incubated by dispensing a samplesolution between the films. The film-type medium provides quantitativeresults in food tests and hence is useful. In environmental monitoringtests, however, bringing the medium into direct contact with a subjectis difficult, unlike the press-type medium, and nutrients or gellingagents that are not firmly bonded are likely to scatter. Otherdisadvantages include leaking of a solution due to degradation andliquefaction of the gel that occur when microorganisms that degrade thegelling agent grow in the medium and liquefy the gel.

As described above, several types of simplified culture media forspecific purposes have been developed and commercially distributed.However, none of them have been applicable to multiple purposes,including food tests and direct contact test of an environment.

Japanese Patent Application Laid-Open No. 6-181741 discloses amicroorganism culture device comprising a laminate of a coldwater-soluble gelling agent, and a water-absorbent fiber sheet. With thecombination of a gelling agent and a water-absorbing fiber sheet in themicroorganism culture device, a portion of an added sample solution isabsorbed by the gelling agent, thereby preventing uniform distributionof the sample solution on the surface of the culture device. Inaddition, the culture device involves a problem in obtainingquantitative results, because microorganisms may fail to form colonieson a top surface of the water-absorbing fiber sheet, and microorganismsgrown on the lower surface of the fiber sheet or inside the sheet aredifficult to count. Therefore, for practical use, this type ofmicroorganism culture device requires a water-absorbing fiber sheet thatis sufficiently thin to be transparent or semitransparent, therebyenabling easy observation of the grown microorganisms. Anotherdisadvantage is that this culture device is not suitable to test methodsinvolving pressing or direct wiping, because the water-absorbing fibersheet and the layer of the cold water-soluble gelling agent are notbonded.

The present inventors have disclosed in International Patent WO 97/24432a sheet- or film-form microorganism culture medium comprising a porousmatrix layer and a water-soluble polymer layer. The culture medium isuseful for various purposes, including normal food tests and tests bywiping, produces small wastes, and can provide quantitative results. Inthis type of medium, a sample solution added into the porous matrixlayer is temporarily retained in the porous matrix layer. Thewater-soluble polymer layer coming in contact with the porous matrixlayer dissolves in water in the retained sample solution, andsimultaneously nutrients for microorganism growth contained in thewater-soluble polymer layer also dissolve in the water, therebyinitiating microorganism growth. When the sample solution is added tothe medium, the microorganisms in the solution disperse in the porousmatrix layer but not inside the water-soluble polymer layer. This isbecause a high-viscosity layer formed by gradual dissolution of thewater-soluble polymer layer surface prevents movement of themicroorganisms into the water-soluble polymer layer. The dissolvedwater-soluble polymer layer and the porous matrix layer assimilated,thereby pressing the microorganisms up to the porous matrix layersurface, where colonies are formed. Therefore, counting ofmicroorganisms is relatively easy despite use of the porous matrixlayer, because colonies are formed on the porous matrix layer surface.

However, despite the excellent features of the sheet- or film-formculture container or medium comprising a porous matrix layer and awater-soluble polymer layer, some drawbacks are involved, due to lack ofoptimal physical properties of the porous matrix layer specifically forculturing microorganisms. For example, some sample solutions exhibitpoor dispensability of microorganisms or a smaller number of observablemicroorganisms growing in the medium as compared with the case of astandard method. In addition, in the case where a significant number ofmicroorganisms are present, growth of the microorganisms may beimpossible to observe. Therefore, prior to culturing microorganisms, auser of this sheet- or film-form culture container or medium mustconsider various factors, including retention of the sample solution,its dispensability, or permeability of the matrix layer to thewater-soluble polymer that is dissolved by the sample solution. Further,in microbial tests involving numerous samples, time-consumingbag-packing of the medium is required after sample addition, in order toprevent unwanted microorganisms from contaminating the medium, and thepacked media are bulky.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a microorganism culturedevice which is suitable to conventional microbial tests for foods andan environmental monitoring and is easily applicable to microbial testscarried out on a curved or uneven surface as well as those carried outon a flat surface, by directly pressing or wiping test subjects, and toa microorganism culture medium comprising the same. A further object ofthe present invention is to provide a sheet-form microorganism culturedevice which is compact, easy to use, and capable of preventingcontamination by unwanted microorganisms, as well as a sheet-formmicroorganism culture medium comprising the same.

The present inventors have conducted thorough research in an effort tosolve the above-described problems and, as a result, have found that theobjects are achieved by the means described below, thus leading toaccomplishment of the present invention. Namely, the present inventionprovides the following.

(1) A microorganism culture device comprising a porous matrix layerhaving a basis weight of 40 to 100 g/m² and an air permeability of 7 to24 cm/sec, and at least one layer of a water-soluble polymer layerlaminated with the matrix layer.

(2) A microorganism culture device according to (1), characterized inthat the water-soluble polymer layer comprises at least two sub-layers.

(3) A microorganism culture device according to (1) or (2),characterized in that when letters are assigned to the sub-layers of thewater-soluble polymer layer in alphabetical order from the sub-layernearest the porous matrix layer, the water-soluble layer comprises twosub-layers, Layer A and Layer B, wherein Layer A contains awater-soluble polymer and Layer B contains or consists of awater-soluble polymer.(4) A microorganism culture device according to (1) or (2),characterized in that when letters are assigned to the sub-layers of thewater-soluble polymer layer in alphabetical order from the sub-layernearest the porous matrix layer, the water-soluble layer comprises threesub-layers, Layer A, Layer B, and Layer C, wherein Layer A and Layer Beach contain a water-soluble polymer and Layer C consists of awater-soluble polymer.(5) A microorganism culture device according to any of (1) to (4),characterized in that the water-soluble polymer in the water-solublepolymer layer contacting the porous matrix layer has a basis weight of 1to 20 g/m².(6) A microorganism culture device according to any of (1) to (5),characterized in that the water-soluble polymer is polyvinyl alcoholhaving a saponification degree of 75 to 95% and a molecular weight of25,000 to 250,000.(7) A microorganism culture device according to any of (1) to (6),characterized in that the porous matrix layer is a nonwoven fabriccomprising at least one type of hydrophilic fiber selected from nylon,rayon, cotton, and cellulose.(8) A microorganism culture device according to (7), characterized inthat the porous matrix layer is a melt-blown nonwoven fabric of nylon.(9) A microorganism culture device according to any of (1) to (8),characterized in that the porous matrix layer has a surface coatinglayer of a water-soluble material.(10) A sheet-form microorganism culture device, characterized in thatthe microorganism culture device according to any of (1) to (9) isbonded at the center of an adhesive sheet larger than the microorganismculture device, with the porous matrix layer being an upper layer and atransparent film larger than the microorganism culture device coveringthe microorganism culture device by contacting the porous matrix layerand aligning the center of the transparent film on the microorganismculture device, wherein a transparent film area that does not contactthe microorganism culture device, and an adhesive sheet area that is notbonded with the microorganism culture device are bonded together.(11) A sheet-form microorganism culture device according to (10),characterized in that the adhesive sheet exhibits adhesion to thetransparent film more strongly in some areas than in the remainingareas.(12) A sheet-form microorganism culture device according to (10),characterized in that the adhesive sheet is partially bonded to thetransparent film with adhesive tapes, which exhibit adhesion strongerthan that of the sheet.(13) A sheet-form microorganism culture device according to any of (10)to (12), characterized in that the adhesive sheet comprises a materialselected from a polyester film having a thickness of 0.07 to 0.5 mm, awhite polyester film, polyolefin-based synthetic paper, andpolyolefin-laminated paper, and has on one side a coating layer of anacrylic adhesive or a rubber adhesive.(14) A sheet-form microorganism culture device according to any of (10)to (13), characterized in that the transparent film is a polyolefin filmor an easily peelable polyolefin film.(15) A sheet-form microorganism culture device according to any of (10)to (14), characterized in that a thickness of the transparent film is 20to 100 μm.(16) A microorganism culture medium, characterized in that themicroorganism culture device according to any of (1) to (9) contains atleast one type of material selected from a nutrient for microorganismgrowth, a coloring agent, and a selective agent.(17) A microorganism culture medium, characterized in that at least onetype of material selected from a nutrient for microorganism growth, acoloring agent, and a selective agent is contained in Layer A and/orLayer B of the microorganism culture device according to (3) to (4).(18) A microorganism culture medium, characterized in that a nutrientfor microorganism growth, a salt, or a mixture thereof is contained inthe water-soluble material according to (9).(19) A microorganism culture medium, characterized in that at least onetype of nutrient for microorganism growth is contained in thewater-soluble polymer layer and/or the porous matrix layer of themicroorganism culture device according to any of (1) to (9).(20) A sheet-form microorganism culture medium, characterized in that atleast one type of material selected from a nutrient for microorganismgrowth, a coloring agent, and a selective agent is contained in thesheet-form microorganism culture device according to any of (10) to(15).(21) A sheet-form microorganism culture medium, characterized in that atleast one type of nutrient for microorganism growth is contained in thewater-soluble polymer layer and/or the porous matrix layer of thesheet-form microorganism culture device according to any of (10) to(15).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph comparing the performance of the culture medium fortotal aerobic counts used in Example 1 and that of a plate count agar.

FIG. 2 is a graph comparing the performance of the culture medium fortotal aerobic counts used in Comparative Example 1 and that of a platecount agar.

FIG. 3 is a schematic illustration of an example of the sheet-formmicroorganism culture device of the present invention.

FIG. 4 is a schematic illustration of an example of the sheet-formmicroorganism culture device of the present invention.

REFERENCE NUMERALS

1: sheet-form microorganism culture device, 2: porous matrix layer, 3:transparent film, 4: adhesive sheet, 5: water-soluble polymer layer

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in detail as follows.

The microorganism culture device of the present invention comprises alaminate of a porous matrix layer having a basis weight of 40 to 100g/m² and an air permeability of 7 to 24 cm/sec, and at least one layerof a water-soluble polymer, wherein the porous matrix layer is laminatedon the water-soluble layer. In addition, the microorganism culturedevice is rarely kept outside during storage or culture, but is normallysealed in a container so as to prevent unwanted microorganisms fromcontaminating the culture device. In the present invention, a sheet-formmicroorganism culture device is provided by bonding the microorganismculture device at the center of an adhesive sheet larger than themicroorganism culture device with the porous matrix layer serving as anupper layer, covering the microorganism culture device with atransparent film larger than the microorganism culture device bycontacting the transparent film and the porous matrix layer and aligningthe center of the transparent film on the microorganism culture device,and bonding a transparent film area that does not contact themicroorganism culture device and an adhesive sheet area that is notbonded with the microorganism culture device. Namely, sealing themicroorganism culture device in a sheet-form container provides a lightweight, compact, and easily handled sheet-form microorganism culturedevice. In the present invention, a microorganism culture devicecontaining nutrients for microorganism growth is defined as amicroorganism culture medium. Similarly, a sheet-form microorganismculture device containing nutrients for microorganism growth is definedas a sheet-form microorganism culture medium. The microorganism culturedevice and the sheet-form microorganism culture device are used asculture media by adding at least one type of nutrient for microorganismgrowth to the culture devices. Methods of adding the nutrient to theculture devices include adding the nutrient to the water-soluble polymerlayer contacting the porous matrix layer in advance in the coursepreparation of the water-soluble polymer layer. Another method is addingon the porous matrix layer of the microorganism culture device a samplesolution containing a nutrient during the course of test. The samplesolution (water or water containing the nutrient for microorganismgrowth) added on the porous matrix layer is temporarily retained in theporous matrix layer, and the water-soluble polymer layer contacting theporous matrix layer is dissolved in the retained water. The resultingaqueous solution of the water-soluble polymer, together with thenutrient, is considered to provide a good environment for microorganismgrowth and initiates fission reproduction of the microorganisms. Whenthe sample solution is added on the porous matrix layer, themicroorganisms in the sample solution uniformly disperse throughout theentire porous matrix layer with the sample solution. The water-solublepolymer slowly dissolves to form a high-viscosity solution, whichcombines the water-soluble polymer layer and the porous matrix layerinto one body. Thus, the microorganisms do not penetrate to the insideof the water-soluble polymer layer, but rather are pushed up to theporous matrix layer surface, around which the microorganisms formcolonies. This mechanism also functions for the microorganism culturedevice packed in a sheet-form container. For the microorganism culturemedium having the porous matrix layer of a basis weight of 40 to 100g/m² and an air permeability of 7 to 24 cm/sec, microorganism growth iseasy to observe even when the number of the microorganisms isconsiderably large, because the fission reproduction occurs on or nearthe surface if the porous matrix layer. Therefore, the colony count isaccurate in the present invention, despite use of a porous matrix layer.

The structures of the microorganism culture device and the sheet-formmicroorganism culture device of the present invention will now bedescribed with reference to FIGS. 3 and 4. The sheet-form microorganismculture device (1) of the present invention comprises the microorganismculture device (5) bonded at the center of an adhesive sheet (4) largerthan the microorganism culture device (5) with a porous matrix layer (2)serving an upper layer, and a transparent film (3) larger than themicroorganism culture device (5) covering the microorganism culturedevice (5) by contacting the porous matrix layer (2) and aligning thecenter of the transparent film (3) on the microorganism culture device(5), wherein a transparent film area that does not contact themicroorganism culture device (5) and an adhesive sheet area that is notbonded with the microorganism culture device (5) are bonded together.

Porous sheets or films are used for the porous matrix layer of thepresent invention. The basis weight of the porous matrix layer ispreferably 50 to 90 g/m², more preferably 55 to 80 g/m². When the basisweight falls significantly below 40 g/m², the porous matrix layer cannotretain added water and sample solutions, possibly resulting in leakageof the sample solutions from the porous matrix layer or inhibition offormation of colonies in an easily observable form, owing toinsufficient fixability of the high-viscosity solution of the dissolvedwater-soluble polymer layer. When the basis weight falls significantlyabove 100 g/m², the high-viscosity solution of the dissolvedwater-soluble polymer layer is retained inside the porous matrix layer,and hence the high-viscosity solution of the dissolved water-solublepolymer layer and the porous matrix layer are insufficiently bonded.This results in the possibility of microorganisms growing inside theporous matrix layer as well as on the surface of the porous matrixlayer, possibly leading to difficulty in observing the microorganismsgrown inside the porous matrix layer and slow growth of themicroorganism near the porous matrix layer surface, due to lack ofnutrition. Air permeability of the porous matrix layer is preferably 7to 24 cm/sec [70 to 240 L/(m²·sec)], more preferably 8 to 20 cm/sec,further preferably 10 to 18 cm/sec. When the air permeability fallssignificantly below 7 cm/sec, water components including a samplesolution fail to be uniformly dispersed in the water-soluble polymerlayer due to lack of water permeability of the porous matrix layer,possibly raising problems, including prevention of uniform culturing ofthe microorganisms. When the air permeability falls significantly above24 cm/sec, the fixability of the high-viscosity solution of thedissolved water-soluble polymer layer becomes insufficient, which islikely to prevent the microorganisms from forming colonies in an easilyobservable form, causing an inaccurate microorganism count.

Fiber materials are used to prepare the porous matrix layer; forexample, synthetic fibers including nylon fibers, polyacrylonitrilefibers, polyvinyl alcohol (PVA) fibers, ethylene-vinyl acetate copolymerfibers, polyester fibers (preferably hydrophilized), polyolefin fibers(preferably hydrophilized), and polyurethane fibers; semi-synthesizedfibers such as rayon fibers; natural fibers including wool (animalfibers), silk, cotton fibers, cellulose fibers, and pulp fibers; andinorganic fibers such as glass fibers. Among these fibers, at least onetype of fiber selected from nylon fibers, cotton fibers, cellulosefibers, and rayon fibers is preferably used for preparing the porousmatrix layer. Porous sheets comprising woven fabrics or nonwoven fabricsof these fibers or porous films, sponges, porous ceramics, etc. madefrom the materials of the fibers listed above can also be used as theporous matrix layer. Porous sheets of woven fabrics or nonwoven fabricswhose basis weight and air permeability are easily adjusted areespecially preferred. Further, melt-blown nylon nonwoven fabricsmanufactured by a melt-blown method, by which fine fibers aremanufactured with relative ease, or super-fine fiber nonwoven fabricsmanufactured from separated fibers, are particularly preferred.

The porous matrix layer of the present invention may be water-repellent.However, a hydrophilic or hydrophilized porous matrix layer ispreferred, because a water absorbing rate is higher than that for thewater-repellent porous matrix layer, and hence the efficiency of thetest work is enhanced. In addition, the surface of the porous matrixlayer is preferably coated with a water-soluble material, because thedispensability of microorganisms is improved. For the water-solublematerial, nutrients for microorganism growth and/or salts are preferablyused, by virtue of their suitability to microorganism culture. Anywater-soluble nutrient for microorganism growth that does not inhibitmicroorganism growth is used as the water-soluble material. Preferredexamples of the water-soluble material include peptones, yeast extract,meat extract, casamino acids, amino acids, amino acid mixtures,saccharides including glucose or maltose, organic acids, and organicacid salts. Salts used for pH control or osmotic pressure control canalso used as the water-soluble material without raising any problem. Forexample, hydrochloric acid salts such as sodium chloride, phosphoricacid salts such as dipotassium phosphate, and carbonic acid salts suchas sodium bicarbonate are preferably used as the water-soluble material.The water-soluble material is coated on the porous matrix layer as asingle component or a mixture. Although the nutrients or the salts canbe sprayed as powders on the porous matrix layer, coating as a solutionor a suspension is preferred. Any solvents that dissolve or partiallydissolve the water-soluble materials can be used to prepare thesolutions or the suspensions and preferable examples are water, ethanol,methanol, and a mixture thereof.

As water-soluble polymers for preparing the water-soluble polymer layerof the present invention, polymers that dissolve in water to exhibit aviscosity of 1×10⁻² Pa·s or greater (at 40° C.) when a sample solutionis added and that do not inhibit microorganism growth are preferablyused. Examples include polyvinyl alcohol, cellulose derivatives,poly(acrylic acid) derivatives, starch derivatives, proteins, proteinderivatives, and polysaccharides. Among these, polyvinyl alcohol is mostpreferable, especially polyvinyl alcohol having a saponification degreeof 75 to 95% and a molecular weight of 25,000 to 250,000.

The microorganism culture device of the present invention contains atleast one water-soluble polymer layer, wherein the water-soluble polymerlayer is either a single layer or multiple layers. The multi-layeredwater-soluble polymer layer is prepared by coating the water-solublepolymer solutions a certain number of times. Alternatively, a certainnumber of water-soluble polymer sub-layers are prepared individually andare subsequently laminated to form the multi-layered water-solublepolymer layer. For example, the microorganism culture device preferablyhas a water-soluble polymer layer comprising Layer A and Layer B whenletters are assigned to the water-soluble polymer sub-layers inalphabetical order from the one nearest the porous matrix layer, whereinLayer A contains a water-soluble polymer and Layer B contains orconsists of a water-soluble polymer. In this case, Layer A preferablycontains at least one type of material selected from a nutrient formicroorganism growth, a coloring agent, and a selective agent, and LayerB either comprises only the water-soluble polymer or additionallycontains at least one type of material selected from a nutrient formicroorganism growth, a coloring agent, and a selective agent. Inanother example, the microorganism culture device preferably has awater-soluble polymer layer comprising Layer A, Layer B, and Layer C,wherein Layer A and Layer B each contain a water-soluble polymer andLayer C consists of a water-soluble polymer. In this case, at least onetype of material selected from a nutrient for microorganism growth, acoloring agent, and a selective agent is preferably contained in Layer Aand/or Layer B. In contrast, Layer C preferably comprises only thewater-soluble polymer. Layer C, which is the water-soluble polymersub-layer furthest from the porous matrix layer, comprising only thewater-soluble polymer reduces the amount of a nutrient for microorganismgrowth to be added to the water-soluble polymer layer, resulting insufficient use of the nutrient for microorganism growth. Incidentally,certain combinations of the coloring agents and the selective agents maycause a reaction when mixed. In order to prevent the reactions, Layer Bis divided into multiple layers and reactive components are addedseparately to different layers. When the water-soluble polymer layer ismulti-layered, the sub-layers are preferably bonded together tightly.Preparing a multi-layered water-soluble polymer layer comprising morethan 5 sub-layers is not sensible, because doing so yields no positiveeffect corresponding to the accompanying increased cost and increasednumber of preparation steps.

In order to use the microorganism culture device of the presentinvention as a microorganism culture medium, a nutrient formicroorganism growth is added to the water-soluble polymer layer inadvance when the microorganism medium is prepared, or water containingthe nutrient for microorganism growth is added when microorganisms areincubated, thus turning the microorganism culture device into a culturemedium. In this case, for the microorganism culture device, favorableculture conditions are established by adding 350 to 650 mL of watercontaining the nutrient for microorganism growth per square meter ofsurface area of the microorganism culture device. Similarly, for themicroorganism culture medium, favorable culture conditions areestablished by adding 350 to 650 mL of water or water containing thenutrient for microorganism growth per square meter of surface area ofthe microorganism culture medium. No particular limitations are imposedon nutrients for microorganism growth used in the present invention, solong as the nutrients are suitable for microorganisms. Examples includea standard liquid culture medium and a culture medium component preparedby removing agar from an agar culture medium. In addition, a selectiveagent for inhibiting growth of non-subject microorganisms, an indicatoror a die for making colonies clearly visible, or a chromogenic orfluorogenic enzyme substrate for detecting a microorganism to bedetermined may be added.

The selective agents of the present invention include antibiotics,synthetic antibacterial agents, pigments, surfactants, and inorganicsalts. Examples of the antibiotics include methicillin, cefmetazole,cefixime, ceftadizime, cefsulodin, bacitracin, polymyxin B, rifampicin,novobiocin, colistin, lincomycin, chloramphenicol, tetracycline, andstreptomycin. Examples of the synthetic antibacterial agents includesulfonamides, nalidixic acid, and olaquindox. Examples of the pigmentsinclude crystal violet, brilliant green, malachite green, and methyleneblue, all of which are bacteriostatic or bactericidal. Examples of thesurfactants include Tergitol 7, dodecylsulfates, and laurylsulfates.Examples of the inorganic salts include selenites, tellurites, sulfites,sodium nitride, lithiumchloride, oxalates, and concentrated sodiumchloride. Further examples of the selective agents includetaurocholates, glycin, bile extract, bile salts, and deoxycholates.

Examples of the nutrients for microorganism growth include, forenumeration of total aerobic counts, a mixture of yeast extract,peptone, and glucose; a mixture of meat extract and peptone; a mixtureof peptone, soybean peptone, and glucose; and any of these mixturesfurther containing dipotassium phosphate and/or sodium chloride.Examples for enumeration of coliforms and/or Escherichia coli include amixture of sodium desoxycholate, peptone, ferric ammonium citrate,sodium chloride, dipotassium phosphate, lactose, and neutral red; and amixture of peptone, lactose, desoxycholate, peptone, eosin Y, andmethylene blue. Examples for test of Staphylococcus aureus include amixture of meat extract, peptone, sodium chloride, manit, phenol red,and yolk; and a mixture of peptone, meat extract, yeast extract, sodiumpyruvate, glycin, lithium chloride, tellurites, and yolk. Examples fortest of vibrio include a mixture of yeast extract, peptone, sucrose,sodium thiosulfate, sodium citrate, sodium cholate, ferric citrate,sodium chloride, ox bile, bromthymol blue, and thymol blue. Examples fortest of enterococci include a mixture of ox brain extract, heartextract, peptone, glucose, dipotassium phosphate, sodium nitride,bromthymol blue, and 2,3,5-triphenyltetrazolium chloride. Examples fortest of fungus include a mixture of peptone and glucose; a mixture ofyeast extract and glucose; and a mixture of potato extract and glucose.

In order to make colonies clearly visible, tetrazolium salts such as2,3,5-triphenyltetrazolium chloride or pH indicators are added to thewater-soluble polymer layer so that a color develops or changes asmicroorganisms grow to thereby visibly indicate the microorganismgrowth. Further, adding coloring or fluorescent enzyme substrates for anenzyme contained in a specified microorganism enables detection of themicroorganism. However, care should be taken in preparing themicroorganism culture device or the microorganism culture medium,because these coloring agents are easily denatured by heat. In addition,tellurites or other components of Vogel-Johnson culture medium orBaird-Parker culture medium for Staphylococcus aureus should be addedonly after other components are sterilized and cooled in preparing themicroorganism culture medium, due to their tendency of thermaldegradation. Antibiotics are also generally prone to thermal degradationand should be added after cooling. When a water-soluble polymer solutionis turned into a film- or sheet-form water-soluble polymer layer, asignificant amount of water should be evaporated, and drying by heat orhot air takes a few minutes. Therefore, for example, in the case wherethe water-soluble polymer solution contains a tetrazolium salt, negativeeffects are caused by the drying process, including coloring of thetetrazolium salt, lowering effects of the tetrazolium salt by itsthermal degradation, or inhibition of microorganism growth by a degradedtetrazolium salt. The water-soluble polymer solution is dried at atemperature sufficiently low and for a sufficiently short time such thatnegative effects of thermal denaturing or thermal degradation are notrevealed if the concentration of the water-soluble polymer solution is 2to 20% and the basis weight of the water-soluble polymer of thewater-soluble polymer layer (content of the water-soluble polymer) is 1to 20 g/m², although feasibility of this method depends on thecomponents. Thus, the microorganism culture device or the microorganismculture medium containing components that are prone to thermaldenaturing or thermal degradation is prepared by preparing in advance awater-soluble polymer layer containing components that are not prone tothermal denaturing or thermal degradation; coating on the layer awater-soluble polymer aqueous solution containing the components thatare prone to thermal denaturing or thermal degradation; laminating theporous matrix layer on the coating; and drying. In the case of thesingle-layered water-soluble polymer layer, the basis weight of thewater-soluble polymer of the water-soluble polymer layer (content of thewater-soluble polymer) that contacts the porous matrix layer ispreferably 1 to 20 g/m², more preferably 3 to 12 g/m², and in the caseof the multi-layered water-soluble polymer layer, the basis weight ispreferably 1 to 20 g/m², more preferably 1 to 10 g/m². When nutrientsfor microorganism growth are added to the water-soluble polymer layer inorder to prepare the microorganism culture medium, the water-solublepolymer of the water-soluble polymer layer turns into a high-viscositysolution by dissolving in an added water solution, resulting in a slowdiffusion rate of the nutrients for microorganism growth and possiblyinsufficient supply to microorganisms of the nutrients for microorganismgrowth. Thus, the nutrients for microorganism growth are likely to beconsumed in insufficient quantity. In order to solve this problem, thereis preferably used the multi-layered water-soluble polymer layer whichcontains a water-soluble polymer sub-layer, furthest from the porousmatrix layer, having no nutrients for microorganism growth, and awater-soluble polymer sub-layer, contacting the porous matrix layer,having the nutrients for microorganism growth, because the nutrients formicroorganism growth are concentrated around the growing microorganismsand high consumption efficiency of the nutrients is achieved. Thewater-soluble polymer sub-layer having no nutrients for microorganismgrowth preferably accounts for 20% or more of the entire water-solublepolymer layer, more preferably 40% or more. Although no upper limit isimposed on this value in terms of function, an upper limit of 80% isimposed when cost and workability are taken into account.

The microorganism culture device or the microorganism culture medium ofthe present invention may further contain a resin film laminated underthe water-soluble polymer layer of the microorganism culture device orthe microorganism culture medium. This resin film is a support film usedin preparing the water-soluble polymer layer and may be removed from thewater-soluble polymer layer as required. The material of the resin filmis at least one type of resin selected from polyesters, polyolefins, andnylons. Use of the resin film comprising these resins is preferred,because adhesion to an adhesive sheet is improved. The microorganismculture device or the microorganism culture medium may be cut into anyshape prior to use.

Any adhesive that does not interfere with microorganism growth is usedfor the adhesive sheets of the present invention. Among such adhesives,an adhesive with sufficiently weak adhesion for re-bonding the sheet andthe transparent film is preferred, in view of good workability. Examplesof such adhesives include rubber adhesives and acrylic adhesives, andpeelable-type and low-adhesion-type acrylic adhesives are particularlypreferable. General purpose thermoplastic resin sheets are used for abase sheet of the adhesive sheet, which needs to be rigid to some extentin order to afford easily peeling of the bonded transparent film yetsufficiently flexible for affording wiping on a curved surface. Examplesinclude polyester films having a thickness of 0.07 to 0.5 mm, whitepolyester films, polyolefin-based synthetic paper, andpolyolefin-laminated paper. The adhesive sheet may be in any shape, butmust have an area that is not bonded with the microorganism culturedevice or the microorganism culture medium around the microorganismculture device, so that the adhesive sheet and the transparent film aretightly bonded at the bonding area with no gap therebetween.

Although any transparent film that does not interfere with microorganismgrowth is used for the transparent film of the present invention,flexible films are preferred, because the microorganism culture deviceor the microorganism culture medium is inserted between the adhesivesheet and the transparent film. Examples thereof include polyolefinfilms, polyester films, nylon films, and polyvinyl(chloride) films.Further, easily peelable films, which are treated with peeling agents,are also preferred, in view of enhancing peelability of the films. Amongthese, polyester films are preferred, and polyolefin films or easilypeelable films are particularly preferred, in terms of workability suchas opening and closing of the transparent films. The thickness of thetransparent film is preferably 20 to 100 μm, in view of flexibility. Thetransparent film may be in any shape but must be larger than themicroorganism culture device or the microorganism culture medium, sothat the adhesive sheet and the transparent film are tightly bonded withno gap therebetween, so as to prevent penetration of unwantedmicroorganisms.

When the transparent film is peeled to open the sheet-form microorganismculture device or the sheet-form microorganism culture medium, thetransparent film may be completely released from the adhesive sheet, butpreferably the transparent film and the adhesive sheet are partiallybonded together, because the transparent film must again cover themicroorganism culture device or the microorganism culture medium andbond with the adhesive sheet. However, the transparent film may beremoved when complete release of the transparent film from thesheet-form microorganism culture device or the sheet-form microorganismculture medium enhances workability in pressing or wiping tests. Theadhesion strength between the transparent film and the adhesive sheet onthe area where the microorganism culture device is not bonded ispreferably stronger than in the remaining area, but sufficiently weak tobe peeled, so that the transparent film is easily peeled to open foraddition of sample solutions with a portion of the film bonded with theadhesive sheet and is bonded to reclose. Further, when the transparentfilm and the adhesive sheet are not completely bonded but have only anarea with stronger adhesion than in the remaining area, the transparentfilm may be removed as required. The transparent film is preferablylarger than the adhesive sheet, in view of easy opening of thetransparent film.

Methods for making an area with strong adhesion in the adhesive sheet ofthe present invention include bonding the transparent film and theadhesive sheet with an adhesive tape having adhesion stronger than thatbetween the adhesive sheet and the transparent film; bonding thetransparent film and the adhesive sheet in one end with a double-sidedadhesive tape; coating adhesives having different adhesion strengths;coating on the transparent film an adhesive having adhesion strongerthan that between the adhesive sheet and the transparent film; andcoating on an area where one end of the adhesive sheet and one end ofthe transparent film overlap an adhesive having adhesion stronger thanthat between the adhesive sheet and the transparent film in theremaining area. When an adhesive tape is used, preferably the adhesivesheet and the transparent film are shifted and the adhesive tape isbonded on both an adhesive side of the adhesive sheet and an area of thetransparent film which does not contact the microorganism culture deviceor the microorganism culture medium, or on both the backside of theadhesive sheet and an area of the transparent film which contacts themicroorganism culture device or the microorganism culture medium. Inanother preferable arrangement, without shifting the adhesive sheet andthe transparent film, the adhesive tape is folded and bonded on both thebackside of the adhesive sheet and an area of the transparent film whichdoes not contact the laminated layers. When the adhesive tape is foldedand bonded on both the backside of the adhesive sheet and an area of thetransparent film which does not contact the microorganism culture deviceor the microorganism culture medium, for the sake of convenience thetransparent film may be completely removed from the adhesive sheet whilethe backside of the adhesive sheet and the transparent film remainbonded. The adhesion of the area with stronger adhesion should bestronger than that of the other area, preferably more than twice asstrong.

Example preparation methods of the microorganism culture device and thesheet-form microorganism culture device of the present invention aredescribed below.

An aqueous solution of a water-soluble polymer (the solution mayoptionally contain nutrients for microorganism growth and may optionallycontain coloring agents or selective agents) is coated on a support filmsuch as a polyester film, to provide a fully dried or partially-driedwater-soluble polymer layer. Additional aqueous solutions ofwater-soluble polymers (the solutions may optionally contain nutrientsfor microorganism growth and may optionally contain coloring agents orselective agents) may be further coated on the water-soluble polymerlayer as required, to form a multi-layered structure. Subsequently, aporous matrix layer is laminated on the water-soluble polymer layer anddried. When the water-soluble polymer layer has a multi-layeredstructure, the top sub-layer, contacting the porous matrix layer, of thewater-soluble polymer layer preferably has a basis weight of 1 to 20g/m². In the processes of laminating the porous matrix layer on thewater-soluble polymer layer and drying, for the single-layeredwater-soluble polymer layer, the porous matrix layer is laminated on apartially-dried water-soluble polymer layer, or the porous matrix layermoistened with water or water containing the nutrients for microorganismgrowth is laminated on a fully dried water-soluble polymer layer. Forthe multi-layered water-soluble polymer layer, the water-soluble polymersolution is over-coated and the porous matrix layer is laminatedimmediately thereafter or after the water-soluble polymer solution ispartially dried. Alternatively, the porous matrix layer is moistenedwith water or water containing the nutrients for microorganism growthand laminated on a fully dried water-soluble polymer layer. After thedrying process, a water-soluble material may be coated on the porousmatrix layer and dried, as required. The support film may be removedfrom the microorganism culture device as required. The microorganismculture device is then cut to a required size, bonded on a sheet or filmcoated with an adhesive, covered with a transparent film, placed in abag or a petri dish, and sterilized with ethylene oxide gas, etc. tothereby prepare a sheet-form microorganism culture device. Themicroorganism culture medium is prepared by adding nutrients formicroorganism growth to the microorganism culture device in the samemanner as for the microorganism culture device. The sheet-formmicroorganism culture medium is also prepared in the same manner. Thewater-soluble polymer layer and the porous matrix layer are preferablybonded at least partially, regardless of the method employed to preparethe microorganism culture device or the microorganism culture medium.

A method for preparing the microorganism culture medium of the presentinvention having three-layered water-soluble polymer layer isexemplified below. The microorganism culture medium and themicroorganism culture device are defined such that the microorganismculture medium contains nutrients for microorganism growth in Layer Aand/or Layer B, whereas the microorganism culture device does notcontain the nutrients for microorganism growth in these layers. Awater-soluble polymer aqueous solution is coated on a resin film, suchas a polyester film, and dried to form a water-soluble polymer sub-layer(Layer C). Another water-soluble polymer aqueous solution containingnutrients for microorganism growth, coloring agents, selective agents,or a mixture thereof is coated on the Layer C and dried to form awater-soluble polymer sub-layer (Layer B). Further, a water-solublepolymer aqueous solution containing nutrients for microorganism growth,coloring agents, or a mixture thereof is coated on Layer B to form awater-soluble polymer sub-layer (Layer A), thereby preparing amulti-layered water-soluble polymer layer comprising Layer C, Layer B,and Layer A. A porous matrix layer which may optionally contain culturemedium components is laminated on the multi-layered water-solublepolymer layer and dried. The microorganism culture medium is prepared bythe above-described processes. In this step, the basis weight of thewater-soluble polymer in the water-soluble polymer sub-layer contactingthe porous matrix layer is preferably 1 to 20 g/m². The porous matrixlayer may be coated with an aqueous solution of nutrients formicroorganism growth including peptone, yeast extract, meat extract,amino acid mixtures, and sugars or salts including sodium chloride,phosphates, and carbonates, or with an aqueous solution of a mixture ofthe nutrients and the salts, after which the aqueous solution is dried.The resin film used as a support film may be removed after the dryingstep, as required. The microorganism culture medium is then cut to anappropriate size and bonded on a larger adhesive sheet. In this step,the adhesive sheet is bonded on the resin film side of the microorganismculture medium when the resin film is not removed, and the adhesivesheet is bonded on the water-soluble polymer layer side when the resinfilm is removed. Subsequently, the microorganism culture medium iscovered with a transparent film and the transparent film is bonded onthe adhesive sheet in the area around the microorganism culture mediumwhere the microorganism culture medium is not bonded. During orsubsequent to this step, adhesion between the adhesive sheet and thetransparent film at one end is made stronger than that between theadhesive sheet and the transparent film in the remaining area byshifting the transparent film and the adhesive sheet at one end andbonding an adhesive tape having adhesion stronger than that between theadhesive sheet and the transparent film; bonding the transparent filmand the adhesive sheet in one end with a double-sided adhesive tapehaving adhesion stronger than that between the adhesive sheet and thetransparent film; or coating on the transparent film, the adhesivesheet, or one end of the film and the sheet an adhesive having adhesionstronger than that of the remaining area. Finally, preparation of thesheet-form microorganism culture medium is completed by sterilizingwith, for example, ethylene oxide gas.

The method for preparing the microorganism culture device or themicroorganism culture medium of the present invention having atwo-layered water-soluble polymer layer is exemplified as follows. Themicroorganism culture medium and the microorganism culture device aredefined such that the microorganism culture medium contains nutrientsfor microorganism growth in Layer A and/or Layer B, whereas themicroorganism culture device does not contain the nutrients formicroorganism growth in these layers. A water-soluble polymer aqueoussolution containing nutrients for microorganism growth, coloring agents,selective agents, or a mixture thereof, or an aqueous solutioncontaining only the water-soluble polymer, is coated on a resin film,such as a polyester film, and dried to form a water-soluble polymersub-layer (Layer B). Another water-soluble polymer aqueous solutioncontaining, as required, nutrients likely to be denatured or decomposedby heat and coloring agents or their mixture, or an aqueous solutioncontaining only the water-soluble polymer, is coated on Layer B to forma water-soluble polymer sub-layer (Layer A), to thereby prepare amulti-layered water-soluble polymer layer comprising Layer B and LayerA. A porous matrix layer which may contain nutrients for microorganismgrowth is laminated on the multi-layered water-soluble polymer layer anddried to thereby prepare the microorganism culture device or themicroorganism culture medium. In this step, the basis weight of thewater-soluble polymer in the water-soluble polymer sub-layer (Layer A)contacting the porous matrix layer is preferably 1 to 20 g/m². The resinfilm used as a support film may be removed after the drying step, asrequired. Subsequently, the microorganism culture device or themicroorganism culture medium is cut to an appropriate size and bonded ona larger adhesive sheet. In this step, the adhesive sheet is bonded onthe resin film side of the microorganism culture device or themicroorganism culture medium when the resin film is not removed, and theadhesive sheet is bonded on the water-soluble polymer layer (Layer B)side when the resin film is removed. Subsequently, the microorganismculture device or the microorganism culture medium is covered with atransparent film, and the transparent film is bonded on the adhesivesheet in the area around or on the microorganism culture medium wherethe microorganism culture device or the microorganism culture medium isnot bonded. During or subsequent to this step, adhesion between theadhesive sheet and the transparent film in one end is made stronger thanthat between the adhesive sheet and the transparent film in theremaining area, by shifting the transparent film and the adhesive sheetin one end and bonding an adhesive tape having adhesion stronger thanthat between the adhesive sheet and the transparent film; bonding thetransparent film and the adhesive sheet in one end with a double-sidedadhesive tape having adhesion stronger than that between the adhesivesheet and the transparent film; or coating on the transparent film, theadhesive sheet, or one end of the film and the sheet an adhesive havingadhesion stronger than that of the remaining area. Finally, preparationof the sheet-form microorganism culture device or the sheet-formmicroorganism culture medium is completed by sterilizing with, forexample, ethylene oxide gas.

Regardless of the method employed to prepare the sheet-formmicroorganism culture device or the sheet-form microorganism culturemedium, the water-soluble polymer sub-layers themselves and thewater-soluble polymer layer and the resin film when the resin film isnot removed are preferably bonded, and the water-soluble polymer layerand the porous matrix layer are preferably at least partially bonded.Preferably, no gap is formed between the transparent film and theadhesive sheet in the bonded area.

The following are example methods of using the sheet-form microorganismculture medium are exemplified.

(1) For microbial test in foods, a sample food and a sterilized salinesolution or sterilized water are homogenized in a sterilized bag with astomacher, and the resulting suspension is diluted to an appropriateconcentration to thereby prepare sample solutions. For environmentalmicrobial monitoring; for example, a food manufacturing environment, asubject surface in a manufacturing site is wiped with a sterilized gauzeor swab, and the gauze, etc., is suspended in a sterilized salinesolution or sterilized water. The resulting suspension is diluted to anappropriate concentration to thereby prepare a sample solution. Thesheet-form microorganism culture device or the sheet-form microorganismculture medium is opened by peeling the transparent film (wherein thearea with stronger adhesion is preferably left bonded), and the samplesolution is added on the porous matrix layer. The microorganism culturemedium is resealed by covering with the transparent film, and isincubated for a predetermined period at an appropriate temperature forculturing microorganisms. Subsequently, measurements includingobservation of microorganism growth and counting of microorganismcolonies are carried out.(2) A sterilized saline solution or sterilized water is added to thesheet-form microorganism culture medium of the present invention, and asubject surface is directly pressed or wiped with the sheet-formmicroorganism culture medium. A sterilized saline solution or sterilizedwater may be added again at this point. The sheet-form microorganismculture medium is covered with a transparent film (plastic films, etc.)so as to prevent evaporation of water, and incubated. Subsequently,measurements including observation of microorganism growth and countingof microorganism colonies are carried out.(3) When a moist surface is tested, pre-moistening the sheet-formmicroorganism culture medium is not required. The surface is directlywiped or pressed with the sheet-form microorganism culture medium, whichis then covered with a transparent film for culturing microorganisms. Asterilized saline solution or sterilized water may be added to thesheet-form microorganism culture medium prior to covering with thetransparent film. Subsequently, measurements including observation ofmicroorganism growth and counting of microorganism colonies are carriedout.(4) The sheet-form microorganism culture medium is left alone after asterilized saline solution or sterilized water is added and fallingmicroorganisms are caught on the sheet-form microorganism culturemedium. Additional sterilized saline solution or sterilized water may beadded at this point. The sheet-form microorganism culture medium iscovered with a transparent film and incubated. Subsequently,measurements including observation of microorganism growth and countingof microorganism colonies are carried out. As described above, the majorfeature of the sheet-form microorganism culture medium lies in itsapplicability to various microbial test methods, including directcontact with a test subject or wiping of a test subject, as well as to aconventional microbial test method for food or an environment. The testmethods by direct contacting or direct wiping are applicable to a testsubject having a curved surface or a surface having some extent ofunevenness. For the sheet-form microorganism culture device, asterilized saline solution or sterilized water containing nutrients formicroorganism growth is used instead of a sterilized saline solution orsterilized water.

EXAMPLES

The present invention is further described in detail with reference tothe following examples, which are not to be construed as restricting thescope of the present invention. The measurement methods employed in thefollowing examples are described below.

(Microorganism Count)

Colonies formed on the microorganism culture medium are counted by eyeobservation.

(Air Permeability)

A porous matrix layer is tested according to a fragile method, a testmethod for nonwoven fabrics for medical use, provided in JIS L 1912(unit: cm/sec).

(Basis Weight)

A porous matrix layer is cut into a square having sides measuring 20 cmand weighed. Subsequently, the weight is converted to weight per 1 m²(unit: g/m²).

(Basis Weight of a Water-Soluble Polymer in a Water-Soluble PolymerLayer)

A water-soluble polymer layer is cut into a square having sidesmeasuring 10 cm, dried, and weighed. Subsequently, the weight isconverted to weight per 1 m² (unit: g/m²). For the water-soluble polymerlayer containing nutrients for microorganism growth, the weight of thenutrients is subtracted, on the basis of a proportional calculation. Forthe multi-layered water-soluble polymer layer, the weight is measuredafter each sub-layer is coated and dried, and the weight differencebetween the steps is calculated.

(Saponification Degree and Polymerization Degree of Polyvinyl Alcohol)

Saponification degree and polymerization degree of polyvinylalcohol aredetermined according to the test method for polyvinyl alcohol providedin JIS K 6726-1994.

(Thickness)

Thickness of sheets and films is measured with Thickness Meter B1 (ToyoSeiki Seisaku-sho, Ltd.).

(Adhesion)

Adhesion is measured with a Tensilon-type tensile testing machine,according to a testing method for adhesive adhesion by de-laminationprovided in JIS K 6854.

Example 1

Polyvinyl alcohol (degree of saponification: 89%, degree ofpolymerization: 1700, molecular weight: approximately 83,000) (45 g),meat extract (1.3 g), peptone (4 g), and dipotassium phosphate (1.3 g)were added to 0.75 L of water and heated for dissolution. The entiretyof the solution was coated on a polyester film having a thickness 20 μm,a width of 0.5 m, and a length of 1 m (support resin film), and dried at110° C. for 15 minutes to thereby prepare a polyvinyl alcohol film(Layer B of the water-soluble polymer layer). Subsequently, polyvinylalcohol (degree of saponification: 89%, degree of polymerization: 1700,molecular weight: approximately 83,000) (5 g), meat extract (0.125 g),peptone (0.4 g), dipotassium phosphate (0.125 g), and2,3,5-triphenyltetrazolium chloride (5 mg) were dissolved in 100 mL ofwater, and the entirety of the solution was coated on Layer B. Amelt-blown nylon nonwoven fabric (porous matrix layer) having a basisweight of 65 g/m² and an air permeability of 12 cm/sec was laminated onthe coating and dried at 100° C. for 30 seconds. The basis weight of thewater-soluble polymer in the polyvinyl alcohol film contacting themelt-blown nylon nonwoven fabric (Layer A of the water-soluble polymerlayer) was 9.8 g/m². The resulting laminate was then cut into a diskhaving a diameter of 50 mm, bonded on the polyester film (support film)side around the center of an adhesive polyester film having a thicknessof 100 μm, a width of 70 mm, and a length of 80 mm; covered with apolypropylene film (transparent film) having a thickness of 0.06 mm, awidth of 70 mm, and a length of 85 mm; and sterilized with ethyleneoxide gas to thereby prepare the sheet-form microorganism culturemedium, which was subsequently used as a culture medium for totalaerobic counts.

Various foods (10 g) including meats, chopped vegetables, and cookedfoods were placed in a sterilized bag, mixed with 100 mL of a sterilizedsaline solution, homogenized in a stomacher, and diluted ten-fold with asterilized saline solution to thereby prepare a sample solution. Theculture medium for total aerobic counts was opened by peeling thepolypropylene film, and 1 mL of the sample solution was added. Theculture medium was reclosed by covering with the polypropylene film, andincubated at 35° C. for 48 hours. Simultaneously, 1 mL of the samplesolution was added in a sterilized petri dish and mixed with a platecount agar, which had been sterilized and stored at 45° C., andmicroorganisms were also incubated in the plate count agar at 35° C. for48 hours. The microorganism counts for both of the media were compared.As shown in FIG. 1, the counts show good correlation, with a correlationcoefficient of 0.999. The microorganism culture medium prepared bylaminating a nonwoven fabric having a basis weight of 65 g/m² and an airpermeability of 10 cm/sec comprising rayon, cotton, and celluloseinstead of the melt-blown nylon nonwoven fabric was also used forsimilar microbial tests, and provided favorable microorganism culture.

Escherichia coli IFO 13500, Bacillus subtilis IFO 3134, Staphylococcusaureus IFO 13276, Enterobacter cloacae JCM 1232, Providenciaalcalifaciens IFO 12931, and Klebsiella oxytoca JCM 1665 were incubatedin a nutrient broth, and the medium was diluted with a sterilized salinesolution to attain a concentration of 10⁴ to 10⁸/mL. The resultingsample solution (1 mL) was added to the prepared culture medium fortotal aerobic counts, and incubated at 35° C. for 48 hours. For all thebacterial strains listed above, uniformly dispersed red spots wereobserved at a sample concentration of 10⁴/mL, and the entire surface ofthe nonwoven fabric turned red at a sample concentration of 10⁵/mL orhigher.

Comparative Example 1

Polyvinyl alcohol (degree of saponification: 89%, degree ofpolymerization: 1700, molecular weight: approximately 83,000) (45 g),meat extract (1.3 g), peptone (4 g), and dipotassium phosphate (1.3 g)were added to 0.75 L of water and heated for dissolution. The entiretyof the solution was coated on a polyester film having a thickness of 20μm, a width of 0.5 m, and a length of 1 m (support resin film), anddried at 110° C. for 15 minutes to thereby prepare a polyvinyl alcoholfilm (water-soluble polymer layer). Subsequently, polyvinyl alcohol(degree of saponification: 89%, degree of polymerization: 1700,molecular weight: approximately 83,000) (5 g), meat extract (0.125 g),peptone (0.4 g), dipotassium phosphate (0.125 g), and2,3,5-triphenyltetrazolium chloride (5 mg) were dissolved in 100 mL ofwater, and the entirety of the solution was coated on the film. Amelt-blown nylon nonwoven fabric (porous matrix layer) having a basisweight of 105 g/m² and an air permeability of 6 cm/sec was laminated onthe coating and dried at 100° C. for 30 seconds. The basis weight of thewater-soluble polymer in the polyvinyl alcohol film (the water-solublepolymer layer) contacting the melt-blown nylon nonwoven fabric was 9.8g/m². The resulting laminate was cut into a disk having a diameter 50mm; bonded on the polyester film (support film) side around the centerof an adhesive polyester film having a thickness 100 μm, a width of 70mm, and a length of 80 mm; covered with a polypropylene film(transparent film) having a thickness of 0.06 mm, a width of 70 mm, anda length of 85 mm; and sterilized with ethylene oxide gas to therebyprepare the sheet-form microorganism culture medium, which wassubsequently used as a culture medium for total aerobic counts.

As in Example 1, various foods (10 g) including meats, choppedvegetables, and cooked foods were placed in a sterilized bag, mixed with100 mL of a sterilized saline solution, homogenized in a stomacher, anddiluted tenfold with a sterilized saline solution to thereby prepare asample solution. The culture medium for total aerobic counts was openedby peeling the polypropylene film, and 1 mL of the sample solution wasadded. The culture medium was reclosed by covering with thepolypropylene film, and incubated at 35° C. for 48 hours.Simultaneously, 1 mL of the sample solution was added in a sterilizedpetri dish and mixed with a plate count agar, which had been sterilizedand stored at 45° C., and also incubated in the plate count agar at 35°C. for 48 hours. The microorganism counts for both of the media werecompared. As shown in FIG. 2, the counts showed fairly good correlation,with a correlation coefficient of 0.949; however, some samples of thesheet-form microorganism culture media showed aerobic count aboutone-tenth those obtained by the plate count agar.

Escherichia coli IFO 13500, Bacillus subtilis IFO 3134, Staphylococcusaureus IFO 13276, Enterobacter cloacae JCM 1232, Providenciaalcalifaciens IFO 12931, and Klebsiella oxytoca JCM 1665 were incubatedin a nutrient broth, and the medium was diluted with a sterilized salinesolution to attain a concentration of 10⁴ to 10⁸/mL. The resultingsample solution (1 mL) was added to the culture medium for total aerobiccounts, and incubated at 35° C. for 48 hours. For Escherichia coli,Enterobacter cloacae, Bacillus subtilis, and Klebsiella oxytoca, colordevelopment was observed at a sample concentration of 10⁶/mL or lower,but no color was observed at a sample concentration of 10⁷/mL or higher.For Staphylococcus aureus, no color was observed at a sampleconcentration of 10⁶/mL or higher, and for Providencia alcalifaciens, nocolor was observed at any sample concentration. The reasons for theseresults are considered to be slow supply of the nutrients formicroorganism growth to the area where the microorganisms were growing,because the dissolved water-soluble polymer failed to reach the nonwovenfabric surface, thereby delaying microorganism growth. Another possiblereason is that the nonwoven fabric interfered with observation of thecolonies.

Example 2

Polyvinyl alcohol (degree of saponification: 89%, degree ofpolymerization: 1700, molecular weight: approximately 83,000) (30 g) wasadded to 0.5 L of water, and heated for dissolution. The entirety of thesolution was coated on a polyester film having a thickness of 20 μm, awidth of 0.5 m, and a length of 1 m (support resin film) and dried at120° C. for 5 minutes to thereby prepare a polyvinyl alcohol film (LayerC of the water-soluble polymer layer). Subsequently, polyvinyl alcohol(degree of saponification: 89%, degree of polymerization: 1700,molecular weight: approximately 83,000) (15 g), peptone (3.75 g),dipotassium phosphate (1 g), lactose (0.2 g), and sodium deoxycholate(0.25 g) were dissolved in 250 mL of water, and the entirety of thesolution was coated on Layer C and dried at 110° C. for 7 minutes tothereby prepare a polyvinyl alcohol film (Layer B of the water-solublepolymer layer). Further, polyvinyl alcohol (degree of saponification:89%, degree of polymerization: 1700, molecular weight approximately83,000) (4 g), peptone (1.25 g), dipotassium phosphate (0.25 g), lactose(0.05 g), and 5-bromo-4-chloro-3-indolyl-□-D-galactopyranoside (25 mg)were dissolved in 100 mL of water, and the entirety of the solution wascoated on Layer B. A melt-blown nylon nonwoven fabric (porous matrixlayer) having a basis weight of 65 g/m² and an air permeability of 10cm/sec was laminated on the coating and dried at 100° C. for 30 seconds.The basis weight of the water-soluble polymer in the polyvinyl alcoholfilm contacting the melt-blown nylon nonwoven fabric (Layer A of thewater-soluble polymer layer) was 7.8 g/m². The resulting laminate wascut into a disk having a diameter 50 mm; bonded on the polyester film(support film) side around the center of an adhesive polyester filmhaving a thickness of 100 μm, a width of 70 mm, and a length of 80 mm;covered with a polypropylene film (transparent film) having a thicknessof 0.06 mm, a width of 70 mm, and a length of 85 mm; and sterilized withethylene oxide gas to thereby prepare the culture medium for coliforms.

Escherichia coli IFO 13500, Klebsiella oxytoca JCM 1665, Klebsiellapneumoniae JCM 1662, Citrobacter freundii IFO 12681, Serratia rubidaeaIFO 12973, and the bacteria strain 1 and 2 isolated from foods wereincubated in a nutrient broth, and the medium was diluted with asterilized saline solution to attain a concentration of 10 to 500cfu/mL. The diluted bacteria sample solution (1 mL) was added to theculture medium for coliforms, and incubated at 36° C. Clearly visibleblue spots were developed for all the bacterial strains after about 16hours.

Comparative Example 2

Polyvinyl alcohol (degree of saponification: 89%, degree ofpolymerization: 1700, molecular weight: approximately 83,000) (45 g),peptone (10 g), dipotassium phosphate (1.2 g), lactose (0.25 g), andsodium deoxycholate (0.3 g) were added to 0.75 L of water and heated fordissolution. The entirety of the solution was coated on a polyester filmhaving a thickness of 20 μm, a width of 0.5 m, and a of length 1 m(support resin film), and dried at 110° C. for 20 minutes to therebyprepare a polyvinyl alcohol film (water-soluble polymer layer).Subsequently, polyvinyl alcohol (degree of saponification: 89%, degreeof polymerization: 1700, molecular weight: approximately 83,000) (4 g),peptone (0.5 g), dipotassium phosphate (0.1 g), lactose (0.025 g), and5-bromo-4-chloro-3-indolyl-□-D-galactopyranoside (25 mg) were dissolvedin 100 mL of water, and the entirety of the solution was coated on thefilm. A melt-blown nylon nonwoven fabric having a basis weight of 65g/m² and an air permeability of 10 cm/sec was laminated on the coatingand dried at 100° C. for 30 seconds. The basis weight of thewater-soluble polymer in the polyvinyl alcohol film (water-solublepolymer layer) contacting the melt-blown nylon nonwoven fabric was 7.8g/m². The resulting laminate was then cut into a disk having a diameter50 mm; bonded on the polyester film (support film) side at around thecenter of an adhesive polyester film having a thickness of 100 μm, awidth of 70 mm, and a length of 80 mm; covered with a polypropylene film(transparent film) having a thickness of 0.06 mm, a width of 70 mm, anda length of 85 mm; and sterilized with ethylene oxide gas to therebyprepare a culture medium for coliforms.

Escherichia coli IFO 13500, Klebsiella oxytoca JCM 1665, Klebsiellapneumoniae JCM 1662, Citrobacter freundii IFO 12681, Serratia rubidaeaIFO 12973, and bacterial strain 1 and 2 isolated from foods wereincubated in a nutrient broth, and the medium was diluted with asterilized saline solution to attain a concentration of 10 to 500cfu/mL. The diluted bacteria sample solution (1 mL) was added to theculture medium for coliforms, and incubated at 36° C. Development ofclearly visible blue spots took about 16 hours for E. coli IFO 13500 andbacteria strain 1 isolated from foods; about 20 hours for K. oxytoca JCM1665 and S. rubidaea IFO 12973; about 25 hours for K. pneumoniae JCM1662 and C. freundii IFO 12681; and about 30 hours for bacterial strain2 isolated from foods.

Example 3

Polyvinyl alcohol (degree of saponification: 89%, degree ofpolymerization: 1700, molecular weight: approximately 83,000) (30 g) wasadded to 0.5 L of water and heated for dissolution. The entirety of thesolution was coated on a polyester film having a thickness of 20 μm, awidth of 0.5 m, and a length of 1 m (support resin film), and dried at120° C. for 5 minutes to thereby prepare a polyvinyl alcohol film (LayerC of the water-soluble polymer layer). Subsequently, polyvinyl alcohol(degree of saponification: 89%, degree of polymerization: 1700,molecular weight: approximately 83,000) (15 g), peptone (3.75 g),dipotassium phosphate (1 g), lactose (0.2 g), and sodium deoxycholate(0.25 g) were dissolved in 0.25 L of water, and the entirety of thesolution was coated on Layer C and dried at 110° C. for 7 minutes tothereby prepare a polyvinyl alcohol film (Layer B of the water-solublepolymer layer). Further, polyvinyl alcohol (degree of saponification:89%, degree of polymerization: 1700, molecular weight: approximately83,000) (5 g), peptone (1.25 g), dipotassium phosphate (0.25 g), lactose(0.05 g), and 5-bromo-4-chloro-3-indolyl-□-D-galactopyranoside servingas a coloring agent (25 mg) were dissolved in 100 mL of water, and theentirety of the solution was coated on Layer B. A melt-blown nylonnonwoven fabric (porous matrix layer) having a basis weight of 65 g/m²and an air permeability of 12 cm/sec was laminated on the coating anddried at 100° C. for 30 seconds to thereby prepare the microorganismculture medium. The polyvinyl alcohol film contacting the melt-blownnylon nonwoven fabric serves as Layer A of the water-soluble polymerlayer. The culture medium was then cut into a disk having a diameter 50mm. A weak adhesion-type acrylic adhesive (manufactured by LintecCorporation) was coated on a white polyester sheet having a thickness of100 μm, a width of 80 mm, and a length of 90 mm to thereby prepare anadhesive sheet, and the disk-shaped microorganism culture medium wasbonded on the polyester film at around the center. Subsequently, theadhesive sheet was covered by a polypropylene film (transparent film)having a thickness of 0.06 mm, a width of 80 mm, and a length of 90 mm,with the polypropylene film shifted by 5 mm in the longitudinaldirection, and the shifted portion was covered by 90 mm-wide bag sealingtape (manufactured by Kyowa Limited Co., Ltd.). The resulting culturemedium was sterilized with ethylene oxide gas to thereby prepare thesheet-form microorganism culture medium for the coliforms. The preparedsheet-form microorganism culture medium was very compact.

When the sheet-form microorganism culture medium was opened by peelingthe polypropylene film (transparent film) from the side opposite the endthat had been bonded with the bag sealing tape, peeling of thepolypropylene film was stopped at the area of 5 mm width where the bagsealing tape had been bonded. Therefore, tasks such as sampling ofmicroorganisms were carried out while the polypropylene film and theadhesive sheet were left bonded.

The sheet-form microorganism culture medium was opened by peeling thepolypropylene film (transparent film) from the side opposite the endthat had been bonded with the bag sealing tape, and 1 mL of a samplesolution, which had been prepared by suspending food wastes in asterilized saline solution and diluting as appropriate, was added to theculture medium. When the peeled polypropylene film was placed back onthe adhesive sheet, the polypropylene film and the adhesive sheet wereeasily re-bonded.

The sheet-form microorganism culture medium was opened by peeling thepolypropylene film (transparent film) from the side opposite the endthat had been bonded with the bag sealing tape until peeling wasstopped, and subsequently the polypropylene film was further peeled morestrongly so to remove the polypropylene film from the adhesive sheetwith the bag sealing tape still bonded on the polypropylene film. After0.4 mL of a sterilized saline solution was added on the porous matrixlayer, a chopping board was wiped with the sheet-form microorganismculture medium. Then, 0.6 mL of a sterilized saline solution was furtheradded, and the peeled polypropylene film was placed back on the adhesivesheet. Although the bag sealing tape was not re-bonded, due to itsdeformation, the polypropylene film was re-bonded to the adhesive sheetwith no gap therebetween. After the sheet-form microorganism culturemedium was incubated at 36° C. for culturing, blue spots were observeddue to the presence of the coliforms, indicating that the sheet-formmicroorganism culture medium is useful as a microorganism culturemedium.

INDUSTRIAL APPLICABILITY

The microorganism culture device and the microorganism culture medium ofthe present invention comprise at least one layer of a water-solublepolymer and a porous matrix layer laminated on the water-soluble polymerlayer, and the porous matrix layer has a basis weight of 40 to 100 g/m²and an air permeability of 7 to 24 cm/sec. By virtue of this structure,no preparation prior to microorganism culture is needed and hence,microbial tests for foods and environments are easily carried out. Inaddition to food tests or environmental tests that are carried outconventionally, subjects having a curved or uneven surface can alsotested by pressing or wiping. Moreover, microorganisms in a samplesolution disperse evenly on the surface of the microorganism culturemedium, and therefore observation of the microorganisms is easy evenwhen the number of the microorganisms is significantly large. Further,because the microorganism culture medium is thin, space required forstorage and culturing is small, and waste is greatly reduced in volumeas compared with the case of a conventional microorganism culturemedium. In addition, because the water-soluble polymer layer ismulti-layered and the basis weight of the water-soluble polymer in thewater-soluble polymer sub-layer contacting the porous matrix layer is 1to 20 g/m², components prone to thermal degradation can be added to thewater-soluble polymer layer. Furthermore, because nutrients formicroorganism growth are not added to the water-soluble polymersub-layer that is furthest from the porous matrix layer when thewater-soluble polymer layer comprises 2 or more sub-layers, the amountof added nutrients that are not taken up by microorganisms is reduced,uptake efficiency of the nutrients is increased, and the preparationprocesses are simplified.

With the sheet-form microorganism culture device and the sheet-formmicroorganism culture medium of the present invention, microbial testsfor foods and environments are easily carried out, and test subjectswith a curved or uneven surface can also be tested by direct pressing orwiping. Because the cover film may be partially bonded or completelyremoved during the course of a test, the most suitable method isselected in accordance with purposes, resulting in simple and easymicrobial tests for various subjects and purposes. Further, because thesheet-form microorganism culture device and the sheet-form microorganismculture medium of the present invention assume the form of a thin sheet,space required for storage is small and waste is greatly reduced involume as compared with the case of conventional microbial tests.

1. A microorganism culture device, comprising: a porous matrix layerhaving a basis weight of 40 to 100 g/m²; air permeability of 7 to 24cm/sec; at least one sub-layer of a multilayered water-soluble polymerlaminated with the matrix layer; and at least one type of materialselected from the group consisting of a nutrient for microorganismgrowth, a coloring agent, and a selective agent, wherein letters areassigned to the sub-layers of the water-soluble polymer layer inalphabetical order beginning with the sub-layer nearest the porousmatrix layer, the water-soluble layer comprising two sub-layers, layer Aand layer B, wherein layer A comprises a water-soluble polymer and layerB comprises a water-soluble polymer; and wherein the at least one typeof material selected from the group consisting of a nutrient formicroorganism growth, a coloring agent, and a selective agent iscontained in layer A, layer B, or in layer A and layer B.
 2. Themicroorganism culture device of claim 1, wherein the porous matrix layerhas a surface coating layer of a water-soluble material, thewater-soluble material further containing a nutrient for microorganismgrowth, a salt, or a mixture thereof.
 3. The microorganism culturedevice of claim 1, wherein at least one nutrient for microorganismgrowth is contained in the water-soluble polymer layer and/or the porousmatrix layer.
 4. A sheet-form microorganism culture device, comprising:a porous matrix layer having a basis weight of 40 to 100 g/m²; airpermeability of 7 to 24 cm/sec; and at least one layer of awater-soluble polymer laminated with the matrix layer, wherein thedevice is bonded at the center of an adhesive sheet larger than thedevice, with the porous matrix layer of the device being an upper layer,and with a transparent film larger than the device covering the deviceby contacting the porous matrix layer and aligning the center of thetransparent film on the device, an area of the transparent film not incontact with the device being bonded to an area of the adhesive sheetnot bonded to the device; and at least one type of material selectedfrom the group consisting of a nutrient for microorganism growth, acoloring agent, and a selective agent.
 5. The sheet-form microorganismculture device of claim 4, wherein at least one nutrient formicroorganism growth is contained in the water-soluble polymer layerand/or the porous matrix layer.
 6. The microorganism culture device ofclaim 1, further comprising a device wherein letters are assigned to thesub-layers of the water-soluble polymer layer in alphabetical orderbeginning with the sub-layer nearest the porous matrix layer, thewater-soluble layer comprising three sub-layers, layers A, B, and C,wherein layer A and layer B comprises a water-soluble polymer and layerC comprises a water-soluble polymer; and wherein the at least one typeof material selected from the group consisting of a nutrient formicroorganism growth, a coloring agent, and a selective agent iscontained in layer A and/or layer B.